The present invention relates to a support system for solid phase synthesis of oligomers, such as oligonucleotides. Furthermore, the invention relates to a method for synthesis of oligonucleotides on a solid support.
Oligonucleotides are polymers built up by polycondensation of ribonucleoside (RNA) or deoxyribonucleoside (DNA) phosphates.
Oligonucleotides can be assembled by repetitive addition of nucleotide monomers using solid-phase methods. Since the introduction of solid-phase synthesis [R. B. Merrifield, J. Am. Cher. Soc. 85 (1963) 2149], the following requirements have been worked out: (1) The solid support must be insoluble and preferably unswellable in the solvent used. (2) Functional groups on the solid support must allow covalent binding of the first nucleoside in a reproducible manner. (3) The solid support must be chemically inert to all reagents used during synthesis and deprotection. The most commonly used supports are controlled pore glass beads (CPG), silica, or polystyrene beads.
Below the synthesis cycle of the commonly used phosphoramidite method is described:
1. Deprotection of the 5xe2x80x2-hydroxyl group in order to generate the parent hydroxyl compounds. This is normally done by treatment of the support with di- or trichloroacetic acid in an organic solvent (for removal of protecting groups).
2. The support is washed in order to remove traces of acid.
3. The 5xe2x80x2-hydroxyl group is reacted with the 3xe2x80x2-phosphoramidite moiety of a properly protected incoming nucleotide (A, C, G or T) in the presence of an activator (e.g. tetrazole) to form a 3xe2x80x2-5xe2x80x2-phosphite triester.
4. Excess reagents are removed by washing with an appropriate solvent.
5. Unreacted 5xe2x80x2-hydroxyl groups are blocked as acetates (capping).
6. The capping reagent is removed by washing.
7. The phosphite triester is then oxidated to the corresponding phosphate triester. This is normally done by the action of aqueous iodine.
8. The oxidation reagents are removed by washing.
The process is repeated until the desired oligonucleotide sequence has been synthesized. After synthesis, all protecting groups are removed and the oligonucleotide is cleaved from the solid support.
In the synthesis, defective oligonucleotides are produced as a consequence of several effects, prominently premature termination of synthesis, followed by capping, which results in 5xe2x80x2 truncated molecules, and depurination during the synthetic cycles that is followed by strand scission during deprotection. Recently, attention has also been directed at the appearance of shorter, internally deleted productsxe2x80x94so called n-1 and n-2 fragments [Temsamani et al, (1995), Nucleic Acids Research 23(11), 1841-1844]; [Fearon et al, (1995) Nucleic acids Res., 23(14), 2754-2761].
The need for pure oligonucleotides is exemplified by the requirement for high quality products in antisense therapy [Gelfi et al, (1996), Antisense and Nucleic Acid Drug Development, 6, 47-53], in routine diagnostics applications, or for physicochemical and structural studies [Agback et al, (1994) Nucleic Acids Res, 22(8), 1404-12]. Also in molecular cloning impure oligonucleotides frequently reduce efficiency and complicate interpretation of results [McClain et al, (1986) Nucleic Acids Res. 14(16), 6770]; [Nassal, (1988) Gene, 66(2), 279-94].
Preparative gel electrophoresis provides the best resolution for purification of oligonucleotides. The method is however laborious, often leading to considerable loss of material, and it is poorly suited for automation and scale-up.
Chromatographic separation can solve some of these problems, offering a potential for scale-up with minimal losses and using fully automatized instruments. These positive aspects are off-set by the rather poor resolving power of most chromatographic systems. As a partial solution to this problem chromatographic separation or oligonucleotides labeled with affinity tags has been used. The commonly used trityl-on oligonucleotide separation on reversed-phase columns, or capture of 5xe2x80x2-thiol labelled or biotinylated oligonucleotides on respective thiol-affinity [Bannwarth et al, (1990), Helv. Chim. Acta, 73, 1139-1147] or avidin columns [Olejnik et al, (1996), 24(2), 361-366] offer the possibility to isolate fragments with intact 5xe2x80x2-ends. However, the 5xe2x80x2 part of depurinated molecules notoriously contaminate oligonucleotides purified by this method.
A mild basic system has been proposed for partial deprotection and cleavage of apurinic-sites with the oligonucleotides still bound to the solid support. In this manner the 5xe2x80x2 ends of depurinated molecules can be discarded before the oligonucleotides are released from the support, followed by isolation of molecules with intact 5xe2x80x2 ends [Horn et al, (1988), Nucleic Acids Res, 16(24), 11559-71]. In practice, this strategy was accompanied bag a substantial loss of products, due to inadvertent release of oligonucleotides during cleavage of depurinate sites.
In WO92/0915 there is described the use of an alkoxysilyl group as a linker of the oligonucleotide to the support.
This linker is inert during the synthetic cycles and it resists conditions that cleave apurinic sites. The linker is finally cleaved from the solid support with tetra butyl ammonium fluoride (TBAF) to obtain, after reversed-phase separation of DMTr-containing material, an oligonucleotide with both 3xe2x80x2- and 5xe2x80x2-ends intact. However, synthesis of this support was laborious and inconvenient. Due to low reactivity of the functional group of the linker the degree of substitution of the support becomes low which leads to insufficient nucleoside loadings of the support. Thus, this method is not suitable for preparation of support useful for large scale synthesis.
According to a first aspect, the invention provides a support system for solid phase synthesis of oligomers. The support system comprises a support, a linker and a starting compound of the oligomer. The starting compound is bound to the support via a disiloxyl linkage. The disiloxyl function is linked to a hydroxyl group on the support. The functional groups connected to the disiloxyl group are very reactive allowing for reproducible and controlled loading of the starting compounds.
The support system, of the invention is easier to produce compared to prior art systems and provides for high loadings to the support. According to the invention high loading values are obtained for the starting nucleoside. These loadings, often higher than 200 xcexcmol/g, are required for cost-effective large scale synthesis.
The linkage is inert during the synthesis cycles and resists conditions that cleave apurinic sites.
In a preferred embodiment, the starting compound is a nucleoside and the solid phase synthesis is used for the synthesis of oligonucleotides.
Supports with immobilized oligonucleotide can be used as hybridization affinity matrices. Some possible applications of such supports are: purification of DNA-binding proteins, affinity purification of plasmids, as a support for gene assembly (from oligonucleotides) and for diagnostic purposes, etc.
In the new support system of the present invention the first nucleoside is bound to the support via a disiloxyl linkage and the system is preferably represented by the following formula (I). 
wherein
B is a ribonucleoside or deoxyribonucleoside base; R2 is xe2x80x94H, xe2x80x94OH, or OR7 in which R7 is a protecting group; R1 is a protecting group; R3, R4, R5, R6 taken separately each represent alkyl, aryl, cycloalkyl, alkenyl, aralkyl, cycloalkylalkyl, alkyloxy, aryloxy, cycloalkyloxy, alkenyloxy and aralkyloxy; Supp is a solid support; X is an anchoring group used for covalent bonding to the support; p-Y)n and p-Z)m are oligophosphotriester linkers, wherein p represents a phosphotriester, Y and Z are independently selected from a nucleoside and a rest of a diol, A is an alifatic or aromatic group, n is a number between 0-50, preferably 0-10, and k, 1, m are each a number of 0 or 1, with the proviso that when m and n are 0 then 1 and k are 0 and with the proviso that when m=1 then k is 0 and X is O or S.
The protecting groups R1 and R7 are protecting groups usually used for protection of 5xe2x80x2 and 2xe2x80x2 position or ribo- and deoxiribonucleosides. R1 may bee selected from a trityl, monomethoxy trityl, dimethoxytrityl, pixyl or other higher alkoxy-substituted trityl-protecting groups. R7 may bee selected from tertbutyldimehylsilyl (TBDMS), methoxytetrahydropyranoyl (MTHP), tetrahydropyranoyl, methyl or allyl.
In a preferred embodiment of the invention R3, R4, R5, R6 are isopropyl. The choice of R3, R4, R5, R6 is dictated by stability vs. lability requirements of the disiloxyl linker. It is known that these properties can easily be controlled by modifying electron donating parameters of the substituents.
X can be any anchoring group, preferably O, S or an amide function, provided it is stable to the conditions used under synthesis and the reagent to cleave apurinic sites. The same proviso applies also to the (p-Y)n linker and (p-Z)m. Thus, there are no other restrictions on Y and Z.
Z is exemplified by a tetraethylene glycol residue.
A wide range of porous as well as non-porous solid supports can be used as supports in methods according to the present invention. The group of preferred supports includes cross linked polystyrenes, silica, polysaccharides, crosslinked polysaccharides and various glasses.
When the oligophosphotriester linker p-Y)n is present in the above formula a support is provided resulting in even greater oligonucleotide purity than without said linker. By preparing the supports through a series of synthetic cycles before addition of the cleavable disiloxyl linker and synthesis of the desired oligonucleotide any nonspecific sites of synthesis will be neutralized. This linker gives improved contact between the starting nucleoside and an incoming reagent and ensures that also oligonucleotides starting from sites not intended for synthesis on the support none the less contribute to the production of the desired oligonucleotide.
According to a second aspect the invention provides a method for oligonucleotide synthesis on a solid support Supp. The method comprises the steps:
(i) preparing a support system as defined above;
(ii) condensation of nucleotides onto the first nucleoside of the support system to synthesize an oligonucleotide;
(iii) removal of all protecting groups on the oligonucleotide exept the 5xe2x80x2-protecting group, and cleavage of apurinic sites formed during acid-catalysed deprotection;
(iv) cleavage of the full length product from the support; and
(v) purification of the oligonucleotide
In step (i) an oligophosphotriester linker p-Y)n is synthesized on the solid support and a starting nucleoside is bound to the linker via a (p-z)m linker and a disiloxyl group.
According to one embodiment of the method, the disiloxyl linkage is cleaved selectively according to known methods (Markiewicz W. T., 1979, Journal of Chemical Reserche (M) 0181-0197) with a compound containing fluoride ions, before step (v). A preferred fluoride containing compound is tetra alkyl ammonium fluoride. Trityl ammonium hydrogene fluoride is also suitable. The purification is performed by reversed phase chromatography, using the 5xe2x80x2-protecting group as an affinity handle.
According to an alternative embodiment of the method according to the invention, step (v) is performed by exonuclease treatment whereby non-protected oligonucleotides will be digested. This embodiment is especially suitable for in situ synthesis where chromatography is not possible.